Determination of the sequence of amino acids in proteins and peptides is typically performed by subjecting a protein or peptide of interest to a method known as an Edman degradation. Edman degradation generally involves three chemical steps. First, an amino acid polymer, such as a protein or peptide, reacts with a suitable coupling agent, such as an isothiocyanate (e.g. phenyl isothiocyanate, PITC), at an amino terminus of the amino acid polymer in the presence of a base, such as N-methyl morpholine, to derivatize the terminal amino acid residue. The amino acid chain is then exposed to a suitable cleaving reagent (e.g. trifluoroacetic acid, TFA), whereby the terminal amino acid derivative is cleaved from the amino acid chain. The terminal amino acid derivative is then converted to a more stable derivative and identified by a suitable means, such as by chromatography (e.g., reverse-phase high pressure liquid chromatography (HPLC), thin layer chromatography or gas chromatography). Thereafter, the amino acid chain, having thus been shortened by one residue, is exposed to additional coupling and cleaving reagents to furnish a subsequent cleaved terminal amino acid derivative for identification, thereby allowing determination of the sequence of amino acids in the amino acid chain.
Edman degradation has typically been performed by passing a liquid or gaseous cleaving reagent across a substrate on which a peptide or protein has been deposited. Where a liquid cleaving reagent is employed, the peptide or protein to be sequenced is covalently attached to a substrate. A first volume of liquid reagent, including the coupling reagent in a buffered solution, is passed across the peptide or protein to derivatize the terminal amino acid residue. A second volume of liquid reagent, including the cleaving reagent, is then passed across the derivatized peptide or protein to cleave the terminal amino acid residue. The residue is then isolated and converted for identification. However, many peptides and proteins cannot be covalently attached to substrates because they do not possess the functional groups which are necessary for attachment. Another disadvantage is that the method generally requires consumption of large volumes of liquid during the coupling and cleaving steps. Often, impurities in the reagents and liquids can lower the efficiency of the process.
A second method of performing the Edman degradation includes depositing a peptide or protein onto a porous substrate by adsorption. Loss of the amino acid chain from the substrate is generally avoided by exposing the amino acid chain to coupling and cleaving reagents in the form of gases which are passed across or through the porous substrate. However, the cleaving step generates fewer cleaved derivatized residues than does the method described above which employs liquid reagents. Thus, the repetitive efficiency of the procedure is significantly diminished. Also, the utility of protocols employing gaseous-phase coupling and cleaving reagents is generally limited to the analysis of proteins and peptides that may be adsorptively bound to a substrate.
Therefore, a method is needed for determining the sequence of proteins and peptides by Edman degradation which overcome the above-mentioned problems.